Antisense oligonucleotide modulation of raf gene expression

ABSTRACT

Oligonucleotides are provided which are targeted to nucleic acids encoding human raf and capable of inhibiting raf expression. The oligonucleotides may have chemical modifications at one or more positions and may be chimeric oligonucleotides. Methods of inhibiting the expression of human raf using oligonucleotides of the invention are also provided. The present invention further comprises methods of inhibiting hyperproliferation of cells and methods of treating or preventing conditions, including hyperproliferative conditions, associated with raf expression.

This application is a continuation-in-part of Ser. No. 10/057,550, filed Jan. 25, 2002, which is a continuation of Ser. No. 09/506,073, filed Feb. 18, 2000, which is a continuation-in-part of Ser. No. 09/143,214 filed Aug. 28, 1998, now issued as U.S. Pat. No. 6,090,626, which is a continuation of Ser. No. 08/756,806 filed Nov. 26, 1996, now issued as U.S. Pat. No. 5,952,229 which was a continuation of PCT/US95/07111 filed May 31, 1995 and Ser. No. 08/250,856 filed May 31, 1994, now issued as U.S. Pat. No. 5,563,255. This application is also a continuation-in-part of Ser. No. 08/888,982, filed Jul. 7, 1997, now issued as U.S. Pat. No. 5,981,731, and corresponding PCT application PCT/US98/13961, filed Jul. 6, 1998. Each of these applications is assigned to the assignee of the present invention.

INTRODUCTION

1. Field of the Invention

This invention relates to compositions and methods for modulating expression of the raf gene, a naturally present cellular gene which has been implicated in abnormal cell proliferation and tumor formation. This invention is also directed to methods for inhibiting hyperproliferation of cells; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the raf gene and to prevention of tumor metastasis.

2. Background of the Invention

Alterations in the cellular genes which directly or indirectly control cell growth and differentiation are considered to be the main cause of cancer. The raf gene family includes three highly conserved genes termed A-, B- and c-raf (also called raf-1). Raf genes encode protein kinases that are thought to play important regulatory roles in signal transduction processes that regulate cell proliferation. Expression of the c-raf protein is believed to play a role in abnormal cell proliferation since it has been reported that 60% of all lung carcinoma cell lines express unusually high levels of c-raf mRNA and protein. Rapp et al., The Oncogene Handbook, E. P. Reddy, A. M Skalka and T. Curran, eds., Elsevier Science Publishers, New York, 1988, pp. 213-253.

Oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. For example, workers in the field have now identified antisense, triplex and other oligonucleotide compositions which are capable of modulating expression of genes implicated in viral, fungal and metabolic diseases. Antisense oligonucleotides have been safely administered to humans and clinical trials of several antisense oligonucleotide drugs, targeted both to viral and cellular gene products, are presently underway. The phosphorothioate oligonucleotide drug, Vitravene™ (ISIS 2922), has been approved by the FDA for treatment of cytomegalovirus retinitis in AIDS patients. It is thus established that oligonucleotides can be useful therapeutic instrumentalities and can be configured to be useful in treatment regimes for treatment of cells and animal subjects, especially humans.

Antisense oligonucleotide inhibition of gene expression has proven to be a useful tool in understanding the roles of raf genes. An antisense oligonucleotide complementary to the first six codons of human c-raf has been used to demonstrate that the mitogenic response of T cells to interleukin-2 (IL-2) requires c-raf. Cells treated with the oligonucleotide showed a near-total loss of c-raf protein and a substantial reduction in proliferative response to IL-2. Riedel et al., Eur. J. Immunol. 1993, 23, 3146-3150. Rapp et al. have disclosed expression vectors containing a raf gene in an antisense orientation downstream of a promoter, and methods of inhibiting raf expression by expressing an antisense Raf gene or a mutated Raf gene in a cell. WO application 93/04170. An antisense oligodeoxyribonucleotide complementary to codons 1-6 of murine c-Raf has been used to abolish insulin stimulation of DNA synthesis in the rat hepatoma cell line H4IIE. Tornkvist et al., J. Biol. Chem. 1994, 269, 13919-13921. WO Application 93/06248 discloses methods for identifying an individual at increased risk of developing cancer and for determining a prognosis and proper treatment of patients afflicted with cancer comprising amplifying a region of the c-raf gene and analyzing it for evidence of mutation.

Denner et al. disclose antisense polynucleotides hybridizing to the gene for raf, and processes using them. WO 94/15645. Oligonucleotides hybridizing to human and rat raf sequences are disclosed.

Iversen et al. disclose heterotypic antisense oligonucleotides complementary to raf which are able to kill ras-activated cancer cells, and methods of killing raf-activated cancer cells. Numerous oligonucleotide sequences are disclosed, none of which are actually antisense oligonucleotide sequences.

The liver is a major site of metastases for some of the most common malignancies, carcinomas of the gastrointestinal tract and colorectal carcinomas in particular. Liver metastases are frequently inoperable and are associated with poor prognosis. New approaches based on an understanding of the biology of liver metastasis may provide alternative strategies for prevention and treatment of hepatic metastases. The metastatic cascade involves a sequence of steps including invasion of local host tissues, entry into the circulation, arrest and adherence in the vascular bed and extravasation into the target organ parenchyma. The evidence suggests that attachment of circulating rumor cells to the vascular endothelium or the target organ may be a key event in regulating extravasation and implicates in this adhesion site-specific microvascular endothelial cell surface molecules and cytokine inducible receptors that are normally involved in inflammation-induced leukocyte adhesion and transmigration. Among the cytokine inducible receptors implicated in leukocyte transmigration and tumor metastasis are the selectins, E-selectin in particular.

E-selectin (CD62E) is a 115 kDa antigen first identified on human umbilical vein endothelial cells stimulated by IL-1.

In vivo, its expression on vascular endothelial cells is induced by proinflammatory cytokines such as IL-1 beta and TNF-alpha. The endothelial cells express type 1 (TNFR60) and type 2 (TNFR80) TNF receptors, but the former is thought to be the major form involved in soluble TNF-alpha-induced cellular responses. Signaling through this receptor appears to involve activation of the p42ERK, p38 MAPK and p54JNK (jun-nh2-terminal kinase) pathways, as well as NF-kappa-B activation and may depend on cooperative signaling between these pathways. Recent studies have implicated the ras and raf kinases which act upstream of the MAPK pathway in transcriptional activation of E-selectin, an activity which may be secondary to a RNF-alpha-induced increase in ceramide production.

The selectins generally bind to sialylated, glycosylated or sulfated glycans on glycoproteins, glycolipids or proteoglycan. The tetrasaccharides sialyl-Lewis^(x) (sLew^(x)) and sialyl-Lewis^(a) (s-Lew^(a)) appear to be recognized by all three selectins, namely L-, P- and E-selectin. Sialyl-Lewis^(x) and sialyl-Lewis^(a) have been identified as markers of progression in several types of carcinomas, particularly carcinomas of the gastrointestinal tract which commonly metastasize to the liver and their level of expression in carcinoma-derived cell lines was shown to positively correlate with metastatic ability in nude mice. In vitro adhesion studies have shown that human colorectal, pancreatic and gastric carcinoma cells utilize sLex and related carbohydrates to adhere to TNF-alpha inducible E-selectin on cultured vascular endothelial cells. Moreover, anti-sLe^(x) and Sle^(a) antibodies and a soluble E-selectin fusion protein blocked metastases of human tumors in nude mice implicating E-selectin in the metastatic process, particularly in metastasis of human colorectal carcinoma cells.

Highly metastatic cells entering the liver can rapidly induce a cytokine cascade involving Kupffer cell-derived TNA-alpha which leads to upregulation of hepatic sinusoidal endothelial E-selectin expression which is followed by upregulation of ICAM-1 and VCAM-1. Using an E-selectin specific monoclonal antibody, it was demonstrated that E-selectin is involved in metastasis formation in this organ.

There remains a long-felt need for improved compositions and methods for inhibiting raf gene expression and for preventing tumor metastasis. The present invention addresses this need.

SUMMARY OF THE INVENTION

The present invention provides oligonucleotides which are targeted to nucleic acids encoding human raf and are capable of inhibiting raf expression. The present invention also provides chimeric oligonucleotides targeted to nucleic acids encoding human raf. The oligonucleotides of the invention are believed to be useful both diagnostically and therapeutically, and are believed to be particularly useful in the methods of the present invention.

The present invention also comprises methods of inhibiting the expression of human raf, particularly the abnormal expression of raf. These methods are believed to be useful both therapeutically and diagnostically as a consequence of the association between raf expression and hyperproliferation. These methods are also useful as tools, for example for detecting and determining the role of raf expression in various cell functions and physiological processes and conditions and for diagnosing conditions associated with raf expression.

The present invention also comprises methods of inhibiting hyperproliferation of cells using oligonucleotides of the invention. These methods are believed to be useful, for example in diagnosing raf-associated cell hyperproliferation. These methods employ the oligonucleotides of the invention. These methods are believed to be useful both therapeutically and as clinical research and diagnostic tools.

DETAILED DESCRIPTION OF THE INVENTION

Malignant tumors develop through a series of stepwise, progressive changes that lead to the loss of growth control characteristic of cancer cells, i.e., continuous unregulated proliferation, the ability to invade surrounding tissues, and the ability to metastasize to different organ sites. Carefully controlled in vitro studies have helped define the factors that characterize the growth of normal and neoplastic cells and have led to the identification of specific proteins that control cell growth and differentiation. The raf genes are members of a gene family which encode related proteins termed A-, B- and c-raf. Raf genes code for highly conserved serine-threonine-specific protein kinases. These enzymes are differentially expressed; c-raf, the most thoroughly characterized, is expressed in all organs and in all cell lines that have been examined. A- and B-raf are expressed in urogenital and brain tissues, respectively. c-raf protein kinase activity and subcellular distribution are regulated by mitogens via phosphorylation. Various growth factors, including epidermal growth factor, acidic fibroblast growth factor, platelet-derived growth factor, insulin, granulocyte-macrophage colony-stimulating factor, interleukin-2, interleukin-3 and erythropoietin, have been shown to induce phosphorylation of c-raf. Thus, c-raf is believed to play a fundamental role in the normal cellular signal transduction pathway, coupling a multitude of growth factors to their net effect, cellular proliferation.

Certain abnormal proliferative conditions are believed to be associated with raf expression and are, therefore, believed to be responsive to inhibition of raf expression. Abnormally high levels of expression of the raf protein are also implicated in transformation and abnormal cell proliferation. These abnormal proliferative conditions are also believed to be responsive to inhibition of raf expression. Examples of abnormal proliferative conditions are hyperproliferative disorders such as cancers, tumors, hyperplasias, pulmonary fibrosis, angiogenesis, psoriasis, atherosclerosis and smooth muscle cell proliferation in the blood vessels, such as stenosis or restenosis following angioplasty. The cellular signaling pathway of which raf is a part has also been implicated in inflammatory disorders characterized by T-cell proliferation (T-cell activation and growth), such as tissue graft rejection, endotoxin shock, and glomerular nephritis, for example.

It has now been found that elimination or reduction of raf gene expression may halt or reverse abnormal cell proliferation. This has been found even in when levels of raf expression are not abnormally high. There is a great desire to provide compositions of matter which can modulate the expression of the raf gene. It is greatly desired to provide methods of detection of the raf gene in cells, tissues and animals. It is also desired to provide methods of diagnosis and treatment of abnormal proliferative conditions associated with abnormal raf gene expression. In addition, kits and reagents for detection and study of the raf gene are desired. “Abnormal” raf gene expression is defined herein as abnormally high levels of expression of the raf protein, or any level of raf expression in an abnormal proliferative condition or state.

The present invention employs oligonucleotides targeted to nucleic acids encoding raf. This relationship between an oligonucleotide and its complementary nucleic acid target to which it hybridizes is commonly referred to as “antisense”. “Targeting” an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the target is a nucleic acid encoding raf; in other words, the raf gene or mRNA expressed from the raf gene. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the oligonucleotide interaction to occur such that the desired effect—modulation of gene expression—will result. Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.

In the context of this invention “modulation” means either inhibition or stimulation. Inhibition of raf gene expression is presently the preferred form of modulation. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression or Western blot assay of protein expression as taught in the examples of the instant application. Effects on cell proliferation or tumor cell growth can also be measured, as taught in the examples of the instant application. “Hybridization”, in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them. “Specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.

In preferred embodiments of this invention, oligonucleotides are provided which are targeted to mRNA encoding c-raf, A-raf and B-raf. In accordance with this invention, persons of ordinary skill in the art will understand that mRNA includes not only the coding region which carries the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5′-untranslated region, the 3′-untranslated region, the 5′ cap region, intron regions and intron/exon or splice junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the coding ribonucleotides. In preferred embodiments, the oligonucleotide is targeted to a translation initiation site (AUG codon) or sequences in the 5′- or 3′-untranslated region of the human c-raf mRNA. The functions of messenger RNA to be interfered with include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing or maturation of the RNA and possibly even independent catalytic activity which may be engaged in by the RNA. The overall effect of such interference with the RNA function is to cause interference with raf protein expression.

The present invention provides oligonucleotides for modulation of raf gene expression. Such oligonucleotides are targeted to nucleic acids encoding raf. Oligonucleotides and methods for modulation of c-raf, A-raf and B-raf are presently preferred; however, compositions and methods for modulating expression of other forms of raf are also believed to have utility and are comprehended by this invention. As hereinbefore defined, “modulation” means either inhibition or stimulation. Inhibition of raf gene expression is presently the preferred form of modulation.

In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term “oligonucleotide” also includes oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. Certain preferred oligonucleotides of this invention are chimeric oligonucleotides. “Chimeric oligonucleotides” or “chimeras”, in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the RNA target) and a region that is a substrate for RNase H cleavage. In one preferred embodiment, a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity, and, usually, a region that acts as a substrate for RNAse H. Affinity of an oligonucleotide for its target (in this case a nucleic acid encoding raf) is routinely determined by measuring the Tm of an oligonucleotide/target pair, which is the temperature at which the oligonucleotide and target dissociate; dissociation is detected spectrophotometrically. The higher the Tm, the greater the affinity of the oligonucleotide for the target. In a more preferred embodiment, the region of the oligonucleotide which is modified to increase raf mRNA binding affinity comprises at least one nucleotide modified at the 2′ position of the sugar, most preferably a 2′-O-alkyl, alkyl-O-alkyl or 2′-fluoro-modified nucleotide. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2′-deoxyoligonucleotides against a given target. The effect of such increased affinity is to greatly enhance antisense oligonucleotide inhibition of raf gene expression. RNAse H is a cellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the efficiency of antisense inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis. In another preferred embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide. Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oligonucleotides which contain at least one phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance.

The oligonucleotides in accordance with this invention preferably are from about 8 to about 50 nucleotides in length. In the context of this invention it is understood that this encompasses non-naturally occurring oligomers as hereinbefore described, having 8 to 50 monomers. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides).

As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thiono-alkylphosphonates, thionoalkylphosphotriesters, and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al. (Science, 1991, 254, 1497-1500).

Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl, O-alkyl-O-alkyl, O-, S-, or N-alkenyl, or O-, S- or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504) i.e., an alkoxyalkoxy group. Further preferred modifications include 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) as described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-β-CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920.

Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C or m5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, those disclosed by Englisch et al. (Angewandte Chemie, International Edition 1991, 30, 613-722), and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications 1993, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, X. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications 1993, CRC Press, Boca Raton, pages 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4, 1053-1059), a thioether, e.g., hexyl-5-tritylthiol (Manoharan at al., Ann. N.Y. Acad. Sci. 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J. 1991, 10, 1111-1118; Kabanov et al., FEBS Lett. 1990, 259, 327-330; Svinarchuk et al., Biochimie 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res. 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan at al., Nucleosides & Nucleotides 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routineer. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling Va.) to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides such as cholesterol-modified oligonucleotides.

It has now been found that certain oligonucleotides targeted to portions of the c-raf mRNA are particularly useful for inhibiting raf expression and for interfering with cell hyperproliferation. Methods for inhibiting c-raf expression using antisense oligonucleotides are, likewise, useful for interfering with cell hyperproliferation. In the methods of the invention, tissues or cells are contacted with oligonucleotides. In the context of this invention, to “contact” tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal.

For therapeutics, methods of inhibiting hyperproliferation of cells and methods of treating abnormal proliferative conditions are provided. The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill in the art. In general, for therapeutics, a patient suspected of needing such therapy is given an oligonucleotide in accordance with the invention, commonly in a pharmaceutically acceptable carrier, in amounts and for periods which will vary depending upon the nature of the particular disease, its severity and the patient's overall condition. The pharmaceutical compositions of this invention may be administered in a number of ways depending upon whether local or systemic treatment is desired, and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal), oral, or parenteral, for example by intravenous drip, intravenous injection or subcutaneous, intraperitoneal, intraocular, intravitreal or intramuscular injection.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

In addition to such pharmaceutical carriers, cationic lipids may be included in the formulation to facilitate oligonucleotide uptake. One such composition shown to facilitate uptake is Lipofectin (BRL, Bethesda Md.).

Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities in order to increase the efficacy of a treatment regimen. In the context of the invention, the term “treatment regimen” is meant to encompass therapeutic, palliative and prophylactic modalities. For example, a patient may be treated with conventional chemotherapeutic agents, particularly those used for tumor and cancer treatment. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, teniposide, cisplatin, carboplatin, topotecan, irinotecan, gemcitabine and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Other drugs such as leucovorin, which is a form of folic acid used as a “rescue” after high doses of methotrexate or other folic acid agonists, may also be administered. In some embodiments, 5-FU and leucovorin are given in combination as an IV bolus with the compounds of the invention being provided as an IV infusion.

Dosing is dependent on severity and responsiveness of the condition to be treated, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be calculated based on EC50's in in vitro and in vivo animal studies. For example, given the molecular weight of compound (derived from oligonucleotide sequence and chemical structure) and an effective dose such as an IC50, for example (derived experimentally), a dose in mg/kg is routinely calculated.

The present invention is also suitable for diagnosing abnormal proliferative states in tissue or other samples from patients suspected of having a hyperproliferative disease such as cancer, psoriasis or blood vessel restenosis or atherosclerosis. The ability of the oligonucleotides of the present invention to inhibit cell proliferation may be employed to diagnose such states. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection and, usually, quantitation of such inhibition. Similarly, the present invention can be used to distinguish raf-associated tumors from tumors having other etiologies, in order that an efficacious treatment regime can be designed.

The oligonucleotides of this invention may also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.

The oligonucleotides of the invention are also useful for detection and diagnosis of raf expression. For example, radiolabeled oligonucleotides can be prepared by ³²P labeling at the 5′ end with polynucleotide kinase. Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2, p. 10.59. Radiolabeled oligonucleotides are then contacted with tissue or cell samples suspected of raf expression and the sample is washed to remove unbound oligonucleotide. Radioactivity remaining in the sample indicates bound oligonucleotide (which in turn indicates the presence of raf) and can be quantitated using a scintillation counter or other routine means. Radiolabeled oligo can also be used to perform autoradiography of tissues to determine the localization, distribution and quantitation of raf expression for research, diagnostic or therapeutic purposes. In such studies, tissue sections are treated with radiolabeled oligonucleotide and washed as described above, then exposed to photographic emulsion according to routine autoradiography procedures. The emulsion, when developed, yields an image of silver grains over the regions expressing raf. Quantitation of the silver grains permits raf expression to be detected.

Analogous assays for fluorescent detection of raf expression can be developed using oligonucleotides of the invention which are conjugated with fluorescein or other fluorescent tag instead of radiolabeling. Such conjugations are routinely accomplished during solid phase synthesis using fluorescently labeled amidites or CPG (e.g., fluorescein-labeled amidites and CPG available from Glen Research, Sterling Va. See 1993 Catalog of Products for DNA Research, Glen Research, Sterling Va., p. 21).

Each of these assay formats is known in the art. One of skill could easily adapt these known assays for detection of raf expression in accordance with the teachings of the invention providing a novel and useful means to detect raf expression.

Oligonucleotide Inhibition of c-Raf Expression

The oligonucleotides shown in Table 1 were designed using the Genbank c-raf sequence HSRAFR (Genbank accession no. x03484; SEQ ID NO: 64), synthesized and tested for inhibition of c-raf mRNA expression in T24 bladder carcinoma cells using a Northern blot assay. All are oligodeoxynucleotides with phosphorothioate backbones.

TABLE 1 Human c-raf Kinase Antisense Oligonucleotides SEQ ID Isis # Sequence (5′ → 3′) Site NO: 5000 TGAAGGTGAGCTGGAGCCAT Coding 1 5074 GCTCCATTGATGCAGCTTAA AUG 2 5075 CCCTGTATGTGCTCCATTGA AUG 3 5076 GGTGCAAAGTCAACTAGAAG STOP 4 5097 ATTCTTAAACCTGAGGGAGC 5′UTR 5 5098 GATGCAGCTTAAACAATTCT 5′UTR 6 5131 CAGCACTGCAAATGGCTTCC 3′UTR 7 5132 TCCCGCCTGTGACATGCATT 3′UTR 8 5133 GCCGAGTGCCTTGCCTGGAA 3′UTR 9 5148 AGAGATGCAGCTGGAGCCAT Coding 10 5149 AGGTGAAGGCCTGGAGCCAT Coding 11 6721 GTCTGGCGCTGCACCACTCT 3′UTR 12 6722 CTGATTTCCAAAATCCCATG 3′UTR 13 6731 CTGGGCTGTTTGGTGCCTTA 3′UTR 14 6723 TCAGGGCTGGACTGCCTGCT 3′UTR 15 7825 GGTGAGGGAGCGGGAGGCGG 5′UTR 16 7826 CGCTCCTCCTCCCCGCGGCG 5′UTR 17 7827 TTCGGCGGCAGCTTCTCGCC 5′UTR 18 7828 GCCGCCCCAACGTCCTGTCG 5′UTR 19 7848 TCCTCCTCCCCGCGGCGGGT 5′UTR 20 7849 CTCGCCCGCTCCTCCTCCCC 5′UTR 21 7847 CTGGCTTCTCCTCCTCCCCT 3′UTR 22 8034 CGGGAGGCGGTCACATTCGG 5′UTR 23 8094 TCTGGCGCTGCACCACTCTC 3′UTR 24

In a first round screen of oligonucleotides at concentrations of 100 nM or 200 nM, oligonucleotides 5074, 5075, 5132, 8034, 7826, 7827 and 7828 showed at least 50% inhibition of c-raf mRNA and these oligonucleotides are therefore preferred. Oligonucleotides 5132 and 7826 (SEQ ID NO: 8 and SEQ ID NO: 17) showed greater than about 90% inhibition and are more preferred. In additional assays, oligonucleotides 6721, 7848, 7847 and 8094 decreased c-raf mRNA levels by greater than 50%. These oligonucleotides are also preferred. Of these, 7847 (SEQ ID NO: 22) showed greater than about 90% inhibition of c-raf mRNA and is more preferred.

Specificity of ISIS 5132 for raf

Specificity of ISIS 5132 for raf mRNA was demonstrated by a Northern blot assay in which this oligonucleotide was tested for the ability to inhibit Ha-ras mRNA as well as c-raf mRNA in T24 cells. Ha-ras is a cellular oncogene which is implicated in transformation and tumorigenesis. ISIS 5132 was shown to abolish c-raf mRNA almost completely with no effect on Ha-ras mRNA levels.

2′-Modified Oligonucleotides

Certain of these oligonucleotides were synthesized with either phosphodiester (P═O) or phosphorothioate (P═S) backbones and were also uniformly substituted at the 2′ position of the sugar with either a 2′-O-methyl, 2′-O-propyl, or 2′-fluoro group. Oligonucleotides are shown in Table 2.

TABLE 2 Uniformly 2′ Sugar-modified c-raf Oligonucleotides SEQ ID ISIS # Sequence Site Modif NO. 6712 TCCCGCCTGTGACATGCATT 3′UTR OMe/P = S 8 8033 CGGGAGGCGGTCACATTCGG 5′UTR OMe/P = S 23 7829 GGTGAGGGAGCGGGAGGCGG 5′UTR OMe/P = S 16 7830 CGCTCCTCCTCCCCGCGGCG 5′UTR OMe/P = S 17 7831 TTCGGCGGCAGCTTCTCGCC 5′UTR OMe/P = S 18 7832 GCCGCCCCAACGTCCTGTCG 5′UTR OMe/P = S 19 7833 ATTCTTAAACCTGAGGGAGC 5′UTR OMe/P = S 5 7834 GATGCAGCTTAAACAATTCT 5′UTR OMe/P = S 6 7835 GCTCCATTGATGCAGCTTAA AUG OMe/P = S 2 7836 CCCTGTATGTGCTCCATTGA AUG OMe/P = S 3 8035 CGGGAGGCGGTCACATTCGG 5′UTR OPr/P = 0 23 7837 GGTGAGGGAGCGGGAGGCGG 5′UTR OPr/P = O 16 7838 CGCTCCTCCTCCCCGCGGCG 5′UTR OPr/P = O 17 7839 TTCGGCGGCAGCTTCTCGCC 5′UTR OPr/P = O  18 7840 GCCGCCCCAACGTCCTGTCG 5′UTR OPr/P = O 19 7841 ATTCTTAAACCTGAGGGAGC 5′UTR OPr/P = O 5 7842 GATGCAGCTTAAACAATTCT 5′UTR OPr/P = O 6 7843 GCTCCATTGATGCAGCTTAA AUG OPr/P = O 2 7844 CCCTGTATGTGCTCCATTGA AUG OPr/P = O 3 9355 CGGGAGGCGGTCACATTCGG 5′UTR 2′F/P = S 23

Oligonucleotides from Table 2 having uniform 2′O-methyl modifications and a phosphorothioate backbone were tested for ability to inhibit c-raf protein expression in T24 cells as determined by Western blot assay. Oligonucleotides 8033, 7834 and 7835 showed the greatest inhibition and are preferred. Of these, 8033 and 7834 are more preferred.

Chimeric Oligonucleotides

Chimeric oligonucleotides having SEQ ID NO: 8 were prepared. These oligonucleotides had central “gap” regions of 6, 8, or 10 deoxynucleotides flanked by two regions of 2′-O-methyl modified nucleotides. Backbones were uniformly phosphorothioate. In Northern blot analysis, all three of these oligonucleotides (ISIS 6720, 6-deoxy gap; ISIS 6717, 8-deoxy gap; ISIS 6729, 10-deoxy gap) showed greater than 70% inhibition of c-raf mRNA expression in T24 cells. These oligonucleotides are preferred. The 8-deoxy gap compound (6717) showed greater than 90% inhibition and is more preferred.

Additional chimeric oligonucleotides were synthesized having one or more regions of 2′-O-methyl modification and uniform phosphorothioate backbones. These are shown in Table 3. All are phosphorothioates; bold regions indicate 2′-O-methyl modified regions.

TABLE 3 Chimeric 2′-O-methyl P = S c-raf oligonucleotides Target SEQ ID Isis # Sequence site NO: 7848 TCCTCCTCCCCGCGGCGGGT 5′UTR 20 7852 TCCTCCTCCCCGCGGCGGGT 5′UTR 20 7849 CTCGCCCGCTCCTCCTCCCC 5′UTR 21 7851 CTCGCCCGCTCCTCCTCCCC 5′UTR 21 7856 TTCTCGCCCGCTCCTCCTCC 5′UTR 25 7855 TTCTCGCCCGCTCCTCCTCC 5′UTR 25 7854 TTCTCCTCCTCCCCTGGCAG 3′UTR 26 7847 CTGGCTTCTCCTCCTCCCCT 3′UTR 22 7850 CTGGCTTCTCCTCCTCCCCT 3′UTR 22 7853 CCTGCTGGCTTCTCCTCCTC 3′UTR 27

When tested for their ability to inhibit c-raf mRNA by Northern blot analysis, ISIS 7848, 7849, 7851, 7856, 7855, 7854, 7847, and 7853 gave better than 70% inhibition and are therefore preferred. Of these, 7851, 7855, 7847 and 7853 gave greater than 90% inhibition and are more preferred.

Additional chimeric oligonucleotides with various 2′ modifications were prepared and tested. These are shown in Table 4. All are phosphorothioates; bold regions indicate 2′-modified regions.

TABLE 4 Chimeric 2′-modified P = S c-raf oligonucleotides SEQ Isis Target Modifi-  ID # Sequence site cation NO: 6720 TCCCGCCTGTGACATGCATT 3′UTR 2′-O—Me 8 6717 TCCCGCCTGTGACATGCATT 3′UTR 2′-O—Me 8 6729 TCCCGCCTGTGACATGCATT 3′UTR 2′-O—Me 8 8097 TCTGGCGCTGCACCACTCTC 3′UTR 2′-O—Me 24 9270 TCCCGCCTGTGACATGCATT 3′UTR 2′-O—Pro 8 9058 TCCCGCCTGTGACATGCATT 3′UTR 2′-F 8 9057 TCTGGCGCTGCACCACTCTC 3′UTR 2′-F 24

Of these, oligonucleotides 6720, 6717, 6729, 9720 and 9058 are preferred. Oligonucleotides 6717, 6729, 9720 and 9058 are more preferred.

Two chimeric oligonucleotides with 2′-O-propyl sugar modifications and chimeric P═O/P═S backbones were also synthesized. These are shown in Table 5, in which italic regions indicate regions which are both 2′-modified and have phosphodiester backbones.

TABLE 5 Chimeric 2′-modified P = S/P = O c-raf oligonucleotides SEQ Target Modifi- ID Isis # Sequence site cation NO: 9271 TCCCGCCTGTGACATGCATT 3′UTR 2′-O—Pro 8 8096 TCTGGCGCTGCACCACTCTC 3′UTR 2′-O—Pro 24

Inhibition of Cancer Cell Proliferation

The phosphorothioate oligonucleotide ISIS 5132 was shown to inhibit T24 bladder cancer cell proliferation. Cells were treated with various concentrations of oligonucleotide in conjunction with lipofectin (cationic lipid which increases uptake of oligonucleotide). A dose-dependent inhibition of cell proliferation was demonstrated, as indicated in Table 6, in which “None” indicates untreated control (no oligonucleotide) and “Control” indicates treatment with negative control oligonucleotide. Results are shown as percent inhibition compared to untreated control.

TABLE 6 Inhibition of T24 Cell Proliferation by ISIS 5132 Oligo conc. None Control 5132  50 nM 0  +9% 23% 100 nM 0  +4% 24% 250 nM 0   10% 74% 500 nM 0   18% 82%

Effect of ISIS 5132 on T24 Human Bladder Carcinoma Tumors

Subcutaneous human T24 bladder carcinoma xenografts in nude mice were established and treated with ISIS 5132 and an unrelated control phosphorothioate oligonucleotide administered intraperitoneally three times weekly at a dosage of 25 mg/kg. In this preliminary study, ISIS 5132 inhibited tumor growth after eleven days by 35% compared to controls. Oligonucleotide-treated tumors remained smaller than control tumors throughout the course of the study.

Antisense Oligonucleotides Targeted to A-raf

It is believed that certain oligonucleotides targeted to portions of the A-raf mRNA and which inhibit A-raf expression will, be useful for interfering with cell hyperproliferation. Methods for inhibiting A-raf expression using such antisense oligonucleotides are, likewise, believed to be useful for interfering with cell hyperproliferation.

The phosphorothioate deoxyoligonucleotides shown in Table 7 were designed and synthesized using the Genbank A-raf sequence HUMARAFIR (Genbank listing x04790; SEQ ID NO: 65).

TABLE 7 Oligonucleotides Targeted to Human A-raf SEQ Isis Target NO: # Sequence site ID 9060 GTC AAG ATG GGC TGA GGT GG 5′ UTR 28 9061 CCA TCC CGG ACA GTC ACC AC Coding 29 9062 ATG AGC TCC TCG CCA TCC AG Coding 30 9063 AAT GCT GGT GGA ACT TGT AG Coding 31 9064 CCG GTA CCC CAG GTT CTT CA Coding 32 9065 CTG GGC AGT CTG CCG GGC CA Coding 33 9066 CAC CTC AGC TGC CAT CCA CA Coding 34 9067 GAG ATT TTG CTG AGG TCC GG Coding 35 9068 GCA CTC CGC TCA ATC TTG GG Coding 36 9069 CTA AGG CAC AAG GCG GGC TG Stop 37 9070 ACG AAC ATT GAT TGG CTG GT 3′ UTR 38 9071 GTA TCC CCA AAG CCA AGA GG 3′ UTR 39 10228 CAT CAG GGC AGA GAC GAA CA 3′ UTR 40

Oligonucleotides ISIS 9061, ISIS 9069 and ISIS 10228 were evaluated by Northern blot analysis for their effects on A-raf mRNA levels in A549, T24 and NHDF cells. All three oligonucleotides decreased A-raf RNA levels in a dose-dependent manner in all three cell types, with inhibition of greater than 50% at a 500 nM dose in all cell types. The greatest inhibition (88%) was achieved with ISIS 9061 and 9069 in T24 cells. These three oligonucleotides (ISIS 9061, 9069 and 10228) are preferred, with ISIS 9069 and 9061 being more preferred.

Identification of Oligonucleotides Targeted to Rat and Mouse c-raf

Many conditions which are believed to be mediated by raf kinase are not amenable to study in humans. For example, tissue graft rejection is a condition which is likely to be ameliorated by interference with raf expression; but, clearly, this must be evaluated in animals rather than human transplant patients. Another such example is restenosis. These conditions can be tested in animal models, however, such as the rat and mouse models used here.

Oligonucleotide sequences for inhibiting c-raf expression in rat and mouse cells were identified. Rat and mouse c-raf genes have regions of high homology; a series of oligonucleotides which target both rat and mouse c-raf mRNA sequence were designed and synthesized, using information gained from evaluation of oligonucleotides targeted to human c-raf. These oligonucleotides were screened for activity in mouse bEND cells and rat A-10 cells using Northern blot assays. The oligonucleotides (all phosphorothioates) are shown in Table 8.

TABLE 8 Oligonucleotides targeted to mouse and rat c-raf Target SEQ ID ISIS # site Sequence NO: 10705 Coding GGAACATCTGGAATTTGGTC 41 10706 Coding GATTCACTGTGACTTCGAAT 42 10707 3′UTR GCTTCCATTTCCAGGGCAGG 43 10708 3′UTR AAGAAGGCAATATGAAGTTA 44 10709 3′UTR GTGGTGCCTGCTGACTCTTC 45 10710 3′UTR CTGGTGGCCTAAGAACAGCT 46 10711 AUG GTATGTGCTCCATTGATGCA 47 10712 AUG TCCCTGTATGTGCTCCATTG 48 11060 5′UTR ATACTTATACCTGAGGGAGC 49 11061 5′UTR ATGCATTCTGCCCCCAAGGA 50 11062 3′UTR GACTTGTATACCTCTGGAGC 51 11063 3′UTR ACTGGCACTGCACCACTGTC 52 11064 3′UTR AAGTTCTGTAGTACCAAAGC 53 11065 3′UTR CTCCTGGAAGACAGATTCAG 54

Oligonucleotides ISIS 11061 and 10707 were found to inhibit c-raf RNA levels by greater than 90% in mouse bEND cells at a dose of 400 nM. These two oligonucleotides inhibited raf RNA levels virtually entirely in rat A-10 cells at a concentration of 200 nM. The IC50 for ISIS 10707 was found to be 170 nM in mouse bEND cells and 85 nM in rat A-10 cells. The IC50 for ISIS 11061 was determined to be 85 nM in mouse bEND cells and 30 nM in rat A-10 cells.

Effect of ISIS-11061 on Endogenous c-raf mRNA Expression in Mice

Mice were injected intraperitoneally with ISIS 11061 (50 mg/kg) or control oligonucleotide or saline control once daily for three days. Animals were sacrificed and organs were analyzed for c-raf mRNA expression by Northern blot analysis. ISIS 11061 was found to decrease levels of c-raf mRNA in liver by approximately 70%. Control oligonucleotides had no effects on c-raf expression. The effect of ISIS 11061 was specific for c-raf; A-raf and G3PDH RNA levels were unaffected by oligonucleotide treatment.

Antisense Oligonucleotide to c-raf Increases Survival in Murine Heart Allograft Model

To determine the therapeutic effects of the c-raf antisense oligonucleotide ISIS 11061 in preventing allograft rejection, this oligonucleotide was tested for activity in a murine vascularized heterotopic heart transplant model. Hearts from C57BI10 mice were transplanted into the abdominal cavity of C3H mice as primary vascularized grafts essentially as described by Isobe et al., Circulation 1991, 84, 1246-1255. Oligonucleotides were administered by continuous intravenous administration via a 7-day Alzet pump. The mean allograft survival time for untreated mice was 7.83±0.75 days (7, 7, 8, 8, 8, 9 days). Allografts in mice treated for 7 days with 20 mg/kg or 40 mg/kg ISIS 11061 all survived at least 11 days (11, 11, 12 days for 20 mg/kg dose and >11, >11, >11 days for the 40 mg/kg dose).

In a pilot study conducted in rats, hearts from Lewis rats were transplanted into the abdominal cavity of ACI rats. Rats were dosed with ISIS 11061 at 20 mg/kg for 7 days via Alzet pump. The mean allograft survival time for untreated rats was 8.86±0.69 days (8, 8, 9, 9, 9, 9, 10 days). In rats treated with oligonucleotide, the allograft survival time was 15.3±1.15 days (14, 16, 16 days).

Effects of Antisense Oligonucleotide Targeted to c-raf on Smooth Muscle Cell Proliferation

Smooth muscle cell proliferation is a cause of blood vessel stenosis, for example in atherosclerosis and restenosis after angioplasty. Experiments were performed to determine the effect of ISIS 11061 on proliferation of A-10 rat smooth muscle cells. Cells in culture were grown with and without ISIS 11061 (plus lipofectin) and cell proliferation was measured 24 and 48 hours after stimulation with fetal calf serum. ISIS 11061 (500 nM) was found to inhibit serum-stimulated cell growth in a dose-dependent manner with a maximal inhibition of 46% and 75% at 24 hours and 48 hours, respectively. An IC50 value of 200 nM was obtained for this compound. An unrelated control oligonucleotide had no effect at doses up to 500 nM.

Effects of Antisense Oligonucleotides Targeted to c-raf on Restenosis in Rats

A rat carotid artery injury model of angioplasty restenosis has been developed and has been used to evaluate the effects on restenosis of antisense oligonucleotides targeted to the c-myc oncogene. Bennett et al., J. Clin. Invest. 1994, 93, 820-828. This model will be used to evaluate the effects of antisense oligonucleotides targeted to rat c-raf, particularly ISIS 11061, on restenosis. Following carotid artery injury with a balloon catheter, oligonucleotides are administered either by intravenous injection, continuous intravenous administration via Alzet pump, or direct administration to the carotid artery in a pluronic gel matrix as described by Bennett et al. After recovery, rats are sacrificed, carotid arteries are examined by microscopy and effects of treatment on luminal cross-sections are determined.

Effects of ISIS 5132 (Antisense Oligodeoxynucleotide Targeted To Human c-raf on Tumor Growth in Human Patients

Two clinical trials were undertaken to test ISIS 5132 on a variety of human tumors. In one study the compound was administered by intravenous infusion over 2 hours. In the other trial the drug was administered by intravenous infusion over 21 days using a continuous pump.

Two patients, both of whom had demonstrated tumor progression with previous cytotoxic chemotherapy, exhibited long-term stable disease in response to ISIS 5132 treatment in the 2-hour infusion study (29 patients evaluated). In these responding patients levels of c-raf expression in peripheral blood cells paralleled clinical response. Six patients showed stabilization of disease of two months or greater in response to ISIS 5132 treatment in the 21-day continuous infusion study (34 patients evaluated). These results are discussed hereinbelow in Examples 13-15.

The invention is further illustrated by the following examples which are illustrations only and are not intended to limit the present invention to specific embodiments.

EXAMPLES Example 1 Synthesis and Characterization of Oligonucleotides

Unmodified DNA oligonucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl phosphoramidites were purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step was increased to 68 seconds and was followed by the capping step. 2′-O-methyl phosphorothioate oligonucleotides were synthesized using 2′-O-methyl β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham Mass.) and the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds. The 3′-base used to start the synthesis was a 2′-deoxyribonucleotide. 2′-O-propyl oligonucleotides were prepared by a slight modification of this procedure.

2′-fluoro phosphorothioate oligonucleotides were synthesized using 5′-dimethoxytrityl-3′-phosphoramidites and prepared as disclosed in U.S. patent application Ser. No. 463,358, filed Jan. 11, 1990, and 566,977, filed Aug. 13, 1990, which are assigned to the same assignee as the instant application and which are incorporated by reference herein. The 2′-fluoro oligonucleotides were prepared using phosphoramidite chemistry and a slight modification of the standard DNA synthesis protocol: deprotection was effected using methanolic ammonia at room temperature.

After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides were purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gel electrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mM Tris-borate buffer, pH 7.0. Oligodeoxynucleotides and their phosphorothioate analogs were judged from electrophoresis to be greater than 80% full length material.

Example 2 Northern Blot Analysis of Inhibition of c-raf mRNA Expression

The human urinary bladder cancer cell line T24 was obtained from the American Type Culture Collection (Rockville Md.). Cells were grown in McCoy's 5A medium with L-glutamine (Gibco BRL, Gaithersburg Md.), supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml each of penicillin and streptomycin. Cells were seeded on 100 mm plates. When they reached 70% confluency, they were treated with oligonucleotide. Plates were washed with 10 ml prewarmed PBS and 5 ml of Opti-MEM reduced-serum medium containing 2.5 μl DOTMA. Oligonucleotide with lipofectin was then added to the desired concentration. After 4 hours of treatment, the medium was replaced with McCoy's medium. Cells were harvested 24 to 72 hours after oligonucleotide treatment and RNA was isolated using a standard CsCl purification method. Kingston, R. E., in Current Protocols in Molecular Biology, (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.), John Wiley and Sons, NY. Total RNA was isolated by centrifugation of cell lysates over a CsCl cushion. RNA samples were electrophoresed through 1.2% agarose-formaldehyde gels and transferred to hybridization membranes by capillary diffusion over a 12-14 hour period. The RNA was cross-linked to the membrane by exposure to UV light in a Stratalinker (Stratagene, La Jolla, Calif.) and hybridized to random-primed ³²P-labeled c-raf cDNA probe (obtained from ATCC) or G3PDH probe as a control. RNA was quantitated using a Phosphorimager (Molecular Dynamics, Sunnyvale, Calif.).

Example 3 Specific Inhibition of c-raf Kinase Protein Expression in T24 Cells

T24 cells were treated with oligonucleotide (200 nM) and lipofectin at T=0 and T=24 hours. Protein extracts were prepared at T=48 hours, electrophoresed on acrylamide gels and analyzed by Western blot using polyclonal antibodies against c-raf (UBI, Lake Placid, N.Y.) or A-raf (Transduction Laboratories, Knoxville, Tenn.). Radiolabeled secondary antibodies were used and raf protein was quantitated using a Phosphorimager (Molecular Dynamics, Sunnyvale Calif.).

Example 4 Antisense Inhibition of Cell Proliferation

T24 cells were treated on day 0 for two hours with various concentrations of oligonucleotide and lipofectin (50 nM oligonucleotide in the presence of 2 μg/ml lipofectin; 100 nM oligonucleotide and 2 μg/ml lipofectin; 250 nM oligonucleotide and 6 μg/ml lipofectin or 500 nM oligonucleotide and 10 μg/ml lipofectin). On day 1, cells were treated for a second time at desired oligonucleotide concentration for two hours. On day 2, cells were counted.

Example 5 Effect of ISIS 5132 on T24 Human Bladder Carcinoma Tumor Xenografts in Nude Mice

5×10⁶ T24 cells were implanted subcutaneously in the right inner thigh of nude mice. Oligonucleotides (ISIS 5132 and an unrelated control phosphorothioate oligonucleotide suspended in saline) were administered three times weekly beginning on day 4 after tumor cell inoculation. A saline-only control was also given. Oligonucleotides were given by intraperitoneal injection. Oligonucleotide dosage was 25 mg/kg. Tumor size was measured and tumor volume was calculated on the eleventh, fifteenth and eighteenth treatment days.

Example 6 Diagnostic Assay for raf-Associated Tumors Using Xenografts in Nude Mice

Tumors arising from raf expression are diagnosed and distinguished from other tumors using this assay. A biopsy sample of the tumor is treated, e.g., with collagenase or trypsin or other standard methods, to dissociate the tumor mass. 5×10⁶ tumor cells are implanted subcutaneously in the inner thighs of two or more nude mice. Antisense oligonucleotide (e.g., ISIS 5132) suspended in saline is administered to one or more mice by intraperitoneal injection three times weekly beginning on day 4 after tumor cell inoculation. Saline only is given to a control mouse. Oligonucleotide dosage is 25 mg/kg. Tumor size is measured and tumor volume is calculated on the eleventh treatment day.

Tumor volume of the oligonucleotide-treated mice is compared to that of the control mouse. The volume of raf-associated tumors in the treated mice are measurably smaller than tumors in the control mouse. Tumors arising from causes other than raf expression are not expected to respond to the oligonucleotides targeted to raf and, therefore, the tumor volumes of oligonucleotide-treated and control mice are equivalent.

Example 7 Detection of raf Expression

Oligonucleotides are radiolabeled after synthesis by ³²P labeling at the 5′ end with polynucleotide kinase. Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2, pg. 11.31-11.32. Radiolabeled oligonucleotides are contacted with tissue or cell samples suspected of raf expression, such as tumor biopsy samples or skin samples where psoriasis is suspected, under conditions in which specific hybridization can occur, and the sample is washed to remove unbound oligonucleotide. Radioactivity remaining in the sample indicates bound oligonucleotide and is quantitated using a scintillation counter or other routine means.

Radiolabeled oligonucleotides of the invention are also used in autoradiography. Tissue sections are treated with radiolabeled oligonucleotide and washed as described above, then exposed to photographic emulsion according to standard autoradiography procedures. The emulsion, when developed, yields an image of silver grains over the regions expressing raf. The extent of raf expression is determined by quantitation of the silver'grains.

Analogous assays for fluorescent detection of raf expression use oligonucleotides of the invention which are labeled with fluorescein or other fluorescent tags. Labeled DNA oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl phosphoramidites are purchased from Applied Biosystems (Foster City, Calif.). Fluorescein-labeled amidites are purchased from Glen Research (Sterling Va.). Incubation of oligonucleotide and biological sample is carried out as described for radiolabeled oligonucleotides except that instead of a scintillation counter, a fluorimeter or fluorescence microscope is used to detect the fluorescence which indicates raf expression.

Example 8 Effect of Oligonucleotide on Endogenous c-raf Expression

Mice were treated by intraperitoneal injection at an oligonucleotide dose of 50 mg/kg on days 1, 2 and 3. On day 4 animals were sacrificed and organs removed for c-raf mRNA assay by Northern blot analysis. Four groups of animals were employed: 1) no oligonucleotide treatment (saline); 2) negative control oligonucleotide ISIS 1082 (targeted to herpes simplex virus; 3) negative control oligonucleotide 4189 (targeted to mouse protein kinase C-α; 4) ISIS 11061 targeted to rodent c-raf.

Example 9 Cardiac Allograft Rejection Model

Hearts were transplanted into the abdominal cavity of rats or mice (of a different strain from the donor) as primary vascularized grafts essentially as described by Isobe et al., Circulation 1991, 84, 1246-1255. Oligonucleotides were administered by continuous intravenous administration via a 7-day Alzet pump. Cardiac allograft survival was monitored by listening for the presence of a second heartbeat in the abdominal cavity.

Example 10 Proliferation Assay Using Rat A-10 Smooth Muscle Cells

A10 cells were plated into 96-well plates in Dulbecco's modified Eagle medium (DMEM)+10% fetal calf serum and allowed to attach for 24 hours. Cells were made quiescent by the addition of DMEM+0.2% dialyzed fetal calf serum for an additional 24 hours. During the last 4 hours of quiescence, cells were treated with ISIS 11061+lipofectin (Gibco-BRL, Bethesda Md.) in serum-free medium. Medium was then removed, replaced with fresh medium and the cells were stimulated with 10% fetal calf serum. The plates were the placed into the incubator and cell growth was evaluated by MTS conversion to formozan (Promega cell proliferation kit) at 24 and 48 hours after serum stimulation. A control oligonucleotide, ISIS 1082 (an unrelated oligonucleotide targeted to herpes simplex virus), was also tested.

Example 11 Rat Carotid Artery Restenosis Model

This model has been described by Bennett et al., J. Clin. Invest. 1994, 93, 820-828. Intimal hyperplasia is induced by balloon catheter dilatation of the carotid artery of the rat. Rats are anesthetized and common carotid artery injury is induced by passage of a balloon embolectomy catheter distended with 20 ml of saline. Oligonucleotides are applied to the adventitial surface of the arterial wall in a pluronic gel solution. Oligonucleotides are dissolved in a 0.25% pluronic gel solution at 4° C. (F127, BASF Corp.) at the desired dose. 100 μl of the gel solution is applied to the distal third of the common carotid artery immediately after injury. Control rats are treated similarly with gel containing control oligonucleotide or no oligonucleotide. The neck wounds are closed and the animals allowed to recover. 14 days later, rats are sacrificed, exsanguinated and the carotid arteries fixed in situ by perfusion with paraformaldehyde and glutaraldehyde, excised and processed for microscopy. Cross-sections of the arteries are calculated.

In an alternative to the pluronic gel administration procedure, rats are treated by intravenous injection or continuous intravenous infusion (via Alzet pump) of oligonucleotide.

Example 12 Additional Oligonucleotides Targeted to Human c-raf Kinase

The oligonucleotides shown in Table 9 were designed using the Genbank c-raf sequence HSRAFR (Genbank accession no. x03484; SEQ ID NO: 64), synthesized and tested for inhibition of c-raf mRNA expression as described in Examples 1 and 2. All are oligodeoxynucleotides with phosphorothioate backbones and all are targeted to the 3′ UTR of human c-raf.

TABLE 9 Human c-raf Kinase Antisense Oligonucleotides Isis # Sequence (5′ → 3′) SEQ ID NO 11459 TTGAGCATGGGGAATGTGGG 55 11457 AACATCAACATCCACTTGCG 56 11455 TGTAGCCAACAGCTGGGGCT 57 11453 CTGAGAGGGCTGAGATGCGG 58 11451 GCTCCTGGAAGACAAAATTC 59 11449 TGTGACTAGAGAAACAAGGC 60 11447 CAAGAAAACCTGTATTCCTG 61 11445 TTGTCAGGTGCAATAAAAAC 62 11443 TTAAAATAACATAATTGAGG 63 Of these, ISIS 11459 and 11449 gave 38% and 31% inhibition of c-raf mRNA levels in this assay and are, therefore, preferred. ISIS 11451, 11445 and 11443 gave 18%, 11% and 7% inhibition of c-raf expression, respectively.

Example 13 Effect of Antisense Oligonucleotide Targeted to c-raf on Patients with Cancer—2 Hour Infusion

Twenty-nine fully evaluable patients with a range of cancer types received ISIS 5132 as a two-hour infusion three times weekly for three weeks. Following a one-week treatment-free interval, treatment was resumed, and maintained as long as the patient remained free of tumor progression or significant toxicity. Doses were escalated from 0.5 to 6.0 mg/kg in cohorts of three patients. The drug was well-tolerated and no patient required dose reduction.

Patients with refractory malignancies received ISIS 5132 at 2-hour intravenous infusion three times weekly for 3 consecutive weeks at one of nine dose levels ranging from 0.5 mg/kg to 6.0 mg/kg. Eligibility required adequate bone marrow function (neutrophils≧1,5000/mm³, hemoglobin ≧9.0 g/dL, and platelets ≧1000,000/mm³), serum creatine <2.0 mg/dL, total bilirubin <2.0 mg/dL, aspartate aminotransferase <2 times upper normal limit (<5 times upper normal limit in the presence of liver metastases), and no prolongation of the prothrombin time (PT) or activated partial thromboplastin time (aPTT). Blood counts and biochemical profiles were performed twice weekly during the first week and once a week thereafter. ISIS 5132 was supplied as a sterile solution in vials containing 1.1 mL or 10.5 mL of phosphate-buffered saline at a concentration of 10 mg/mL. Prior to administration, ISIS 5132 was diluted in normal saline to a total volume of 50 mL and the infused intravenously over two hours. Following a one-week treatment-free interval, dosing was resumed and maintained as long as the patient remained free of tumor progression or significant toxicity.

Example 14 Reduction of c-raf Expression in Peripheral Blood Mononuclear Cells of Cancer Patients after Treatment with Antisense Oligonucleotide

Peripheral-blood mononuclear cells (PBMCs) for c-raf mRNA analysis were collected at baseline and on days 3, 5, 8, and 15 of cycle 1 and on day 1 of each cycle thereafter. PBMCs were isolated by Ficoll-Hypaque density centrifugation and stored at −70° C. Total RNA was isolated using Trizol reagent (Gibco BRL, Rockville, Md.) according to the manufacturer's directions. Because of the low abundance of the c-raf message in PBMCs, mRNA quantitation was performed using a reverse-transcriptase polymerase chain reaction (RT-PCR) assay. 100 ng total RNA was used for each cDNA reaction. C-raf expression was normalized to that of the endogenous standard β-actin by calculating the ration of the radiolabeled PCR products. PCR reactions (25 μl total volume, containing 0.1-10 μl cDNA, 12.5 μmol of each of the c-raf or β-actin primers, and 1 μCi α-³²P dCTP) were heated to 95° C. for 5 minutes then amplified for 28-36 cycles at 95° C. for 1 minute, 55° C. for 1 minute and 72° C. for 2 minutes. The products were loaded on 8% urea polyacrylamide gels which were then dried at 80° C. for 1 hour under vacuum and exposed to film for several hours at −80° C. Reductions in c-raf expression were identified in 13 of 14 patients within 48 hours of initial ISIS 5132 dosing. The median reduction was to 42% (mean 53%) of initial values (p=0.002). Compared to baseline values, median reduction in expression on day 5 was 26% (mean 71%; p=0.017), on day 8 32% (mean 81%; p=0.03), and on day 15 35% (mean 74%; p=0.017).

Clinical Responses in Cancer Patients—2 hr Infusion:

Two patients, both of whom had demonstrated tumor progression with previous cytotoxic chemotherapy, exhibited long-term stable disease in response to ISIS 5132 treatment. One was a 68-year old man with colorectal cancer metastatic to liver who had progressed two years after adjuvant therapy with 5-fluorouracil/leucovorin, and had evinced further tumor growth during therapy with a 17-1A monoclonal antibody and irinotecan. Following treatment with 3 mg/kg of ISIS 5132, minor (20%) shrinkage in a liver metastasis was accompanied by a progressive decline in choreoembryonic antigen (CEA, a marker for colon cancer) from 895 ng/mL to 618 ng/mL. During this time, c-raf mRNA values declined to below 10% of the initial value. After seven cycles of treatment, both the plasma CEA values and the PBMC c-raf mRNA began to increase, and one month later a CT scan revealed progression of the hepatic metastases.

A 46-year old woman with renal cell cancer metastatic to lung and lymph nodes failed to respond to interleukin-2, α-interferon and 5-fluorouracil in combination, and began treatment with ISIS 5132 at 5 mg/kg. She had immediate symptomatic improvement, but the size of the tumor was unchanged on CT scans. After ten cycles of treatment, she began to have recurrent pain, and progression was identified radiologically. In this patient the nadir PBMC c-raf mRNA was 9%, and values remained low until the beginning of the ninth cycle, when a return above baseline was observed, again followed shortly thereafter by progressive disease.

Example 15 Effect of Antisense Oligonucleotide Targeted to c-raf on Patients with Cancer—21 Day Continuous Infusion

A continuous intravenous infusion of ISIS 5132 was administered for 21 days every 4 weeks to 34 patients with a variety of solid tumors refractory to standard therapy. The dose of ISIS 5132 was increased in sequential cohorts of patients, as toxicity allowed, until a final dose of 5.0 mg/kg of body weight was reached.

Eligible patients had histologically-documented solid malignancies of measurable or evaluable status refractory to standard therapy or for whom no effective therapy existed. Patients were prescreened in regard to their medical history as described above with the addition of the measurement of complement split products prior to the first infusion of ISIS 5132, 4 and 24 hours after starting the infusion and, repeated on days 7, 14 and 21. Patients received sequential, ascending, multiple doses of ISIS 5132 administered as a continuous IV infusion for 21 consecutive days at a pump rate of 1.5 mL/hour followed by one week of rest (one cycle). The initial dose of ISIS 5231 was 0.5 mg/kg of body weight. Subsequent doses were 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mg/kg. The total dose was added to 250 mL of normal saline and infused as described above.

Clinical Responses in Cancer Patients—22-Day Infusion:

Six patients showed stabilization of disease of two months or greater. Of these two patients had prolonged stabilization: one patient (treated at 1.5 mg/kg/day) with renal cell carcinoma remained stable for 9 months, and the other (treated at 4.0 mg/kg/day) with pancreatic cancer remained stable for 10 months. The most significant response occurred in a 57-year old female with ovarian cancer, treated at 3.0 mg/kg/day. Her CA-125 level (a marker for ovarian cancer) at the time of initial surgical resection was 3300 u/mL. Following resection and a brief course of taxol and platinum, her CA-125 level was reportedly normal, but began to markedly increase again within 8 months. She was then treated with a succession of systemic therapies, most of which achieved only a short term, modest decrease in CA-125 levels. At the time of initiation of ISIS 5132 infusions, her CA-125 level was 1490 u/mL. She was treated with 10 cycles of ISIS 5132 and achieved a 97% reduction in tumor marker levels.

Example 16 Effect of Antisense Oligonucleotide Targeted to c-raf (21 Day Infusion) in Combination with Other Chemotherapeutic Agents in Cancer Patients

Fourteen patients with refractory cancers were given ISIS 5132 at doses of 1.0-3.0 mg/kg/day as a 21 day IV infusion in combination with 5-fluorouracil (425 mg/m²) and Leucovorin (20 mg/m²) as an IV bolus given on days 1-5 every 4 weeks. In this ongoing study, 8 patients have been treated at the 2.0 mg/kg/day dose level. Toxicities that occurred were not dose-limiting. Disease stabilization lasting at least 4 cycles occurred in 4 patients (2 renal cell, 1 colon, 1 pancreatic). Thus ISIS 5132 at a dose of 2 mg/kg/day is active and well tolerated in combination with 5-FU/LV on this schedule.

Example 17 Effect of Antisense Oligonucleotide Targeted to c-raf in Pig Branch Retinal Vein Occlusion Model of Ocular Neovascularization

Angiogenesis, or neovascularization, is the formation of new capillaries from existing blood vessels. In adult organisms this process is typically controlled and short-lived, for example in wound repair and regeneration. Gaiso, M. L., 1999, Medscape Oncology 2(1), Medscape Inc. However, aberrant capillary growth can occur and this uncontrolled growth plays a causal and/or supportive role in many pathologic conditions such as tumor growth and metastasis. In the context of this invention “aberrant angiogenesis” refers to unwanted or uncontrolled angiogenesis. Angiogenesis inhibitors are being evaluated for use as antitumor drugs. Other diseases and conditions associated with angiogenesis include arthritis, cardiovascular diseases, skin conditions, and aberrant wound healing. Aberrant angiogenesis can also occur in the eye, causing loss of vision. Examples of ocular conditions involving aberrant angiogenesis include macular degeneration, diabetic retinopathy and retinopathy of prematurity. A pig model of ocular neovascularization, the branch retinal vein occlusion (BVO) model, is used to study ocular neovascularization. An antisense oligonucleotide targeted to pig c-raf, ISIS 107189 (CCACACCACTCATCTCATCT; SEQ ID NO: 66) was tested in this model.

Male farm pigs (8-10 kg) were subjected to branch retinal vein occlusions (BVO) by laser treatment in both eyes. The extent of BVO was determined by indirect opthalmoscopy after a 2 week period. Intravitreous injections (10 μM) of ISIS 107189 were started on the day of BVO induction and were repeated at weeks 2, 6, and 10 after BVO (Right eye—vehicle, Left eye—antisense molecule). Stereo fundus photography and fluorescein angiography were performed at baseline BVO and at weeks 1, 6 and 12 following intravitreous injections. In addition capillary gel electrophoresis analysis of the eye sections containing sclera, choroid, and the retina were performed to determine antisense concentrations, and gross and microscopic evaluations were performed to determine eye histopathology.

The antisense oligonucleotide targeted to c-raf significantly inhibited the neovascularization response compared to vehicle-only injections (p=0.05).

Example 18 Oligonucleotide Inhibition of B-raf Expression

The oligonucleotides shown in Table 10 were designed using the Genbank B-raf sequence HUMBRAF (Genbank listings M95712; M95720; x54072), provided herein as SEQ ID NO: 67, synthesized and tested for inhibition of B-raf mRNA expression in T24 bladder carcinoma cells or A549 lung carcinoma cells using a Northern blot assay.

The human urinary bladder cancer cell line T24 and the human lung tumor cell line A549 were obtained from the American Type Culture Collection (Rockville Md.). T24 cells were grown in McCoy's 5A medium with L-glutamine and A549 cells were grown in DMEM low glucose medium (Gibco BRL, Gaithersburg Md.), supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml each of penicillin and streptomycin. Cells were seeded on 100 mm plates. When they reached 70% confluency, they were treated with oligonucleotide. Plates were washed with 10 ml prewarmed PBS and 5 ml of Opti-MEM reduced-serum medium containing 2.5 μl DOTMA per 100 nM oligonucleotide. Oligonucleotide with lipofectin was then added to the desired concentration. After 4 hours of treatment, the medium was replaced with appropriate medium (McCoy's or DMEM low glucose). Cells were harvested 24 to 72 hours after oligonucleotide treatment and RNA was isolated using a standard CsCl purification method. Kingston, R. E., in Current Protocols in Molecular Biology. (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.), John Wiley and Sons, NY. Total RNA was isolated by centrifugation of cell lysates over a CsCl cushion. RNA samples were electrophoresed through 1.2% agarose-formaldehyde gels and transferred to hybridization membranes by capillary diffusion over a 12-14 hour period. The RNA was cross-linked to the membrane by exposure to UV light in a Stratalinker (Stratagene, La Jolla, Calif.) and hybridized to a ³²P-labeled B-raf cDNA probe or G3PDH probe as a control. The human B-raf cDNA probe was cloned by PCR using complementary oligonucleotide primers after reverse transcription of total RNA. Identity of the B-raf cDNA was confirmed by restriction digestion and direct DNA sequencing. NA was quantitated using a Phosphorimager (Molecular Dynamics, Sunnyvale, Calif.).

TABLE 10 Human B-raf Kinase Antisense Oligonucleotides (All are phosphorothioate oligodeoxynucleotides) Isis SEQ ID # Sequence (5′ → 3′) Site NO: 13720 ATTTTGAAGGAGACGGACTG coding 68 13721 TGGATTTTGAAGGAGACGGA coding 69 13722 CGTTAGTTAGTGAGCCAGGT coding 70 13723 ATTTCTGTAAGGCTTTCACG coding 71 13724 CCCGTCTACCAAGTGTTTTC coding 72 13725 AATCTCCCAATCATCACTCG coding 73 13726 TGCTGAGGTGTAGGTGCTGT coding 74 13727 TGTAACTGCTGAGGTGTAGG coding 75 13728 TGTCGTGTTTTCCTGAGTAC coding 76 13729 AGTTGTGGCTTTGTGGAATA coding 77 13730 ATGGAGATGGTGATACAAGC coding 78 13731 GGATGATTGACTTGGCGTGT coding 79 13732 AGGTCTCTGTGGATGATTGA coding 80 13733 ATTCTGATGACTTCTGGTGC coding 81 13734 GCTGTATGGATTTTTATCTT coding 82 13735 TACAGAACAATCCCAAATGC coding 83 13736 ATCCTCGTCCCACCATAAAA coding 84 13737 CTCTCATCTCTTTTCTTTTT coding 85 13738 GTCTCTCATCTCTTTTCTTT coding 86 13739 CCGATTCAAGGAGGGTTCTG coding 87 13740 TGGATGGGTGTTTTTGGAGA coding 88 13741 CTGCCTGGATGGGTGTTTTT coding 89 14144 GGACAGGAAACGCACCATAT coding 90 14143 CTCATTTGTTTCAGTGGACA stop codon 91 14142 TCTCTCACTCATTTGTTTCA stop codon 92 14141 ACTCTCTCACTCATTTGTTT stop codon 93 14140 GAACTCTCTCACTCATTTGT coding 94 14139 TCCTGAACTCTCTCACTCAT coding 95 14138 TTGCTACTCTCCTGAACTCT coding 96 14137 TTTGTTGCTACTCTCCTGAG coding 97 14136 CTTTTGTTGCTACTCTCCTG coding 98 13742 GCTACTCTCCTGAACTCTCT coding 99 14135 TTCCTTTTGTTGCTACTCTC coding 100 14134 ATTTATTTTCCTTTTGTTGC coding 101 14133 ATATGTTCATTTATTTTCCT coding 102 13743 TTTATTTTCCTTTTGTTGCT coding 103 13744 TGTTCATTTATTTTCCTTTT coding 104 14132 ATTTAACATATAAGCAAACA coding 105 14529 CTGCCTGGTACCCTGTTTTT 5 mismatch 106 14530 CTGCCTGGAAGGGTGTTTTT 1 mismatch 107 14531 CTGCCTGGTACGGTGTTTTT 3 mismatch 108

There are multiple B-raf transcripts. The two most prevalent transcripts were quantitated after oligonucleotide treatment. These transcripts run at approximately 8.5 kb (upper transcript) and 4.7 kb (lower transcript) under the gel conditions used. Both transcripts are translated into B-raf protein in cells. In the initial screen, A549 cells were treated with oligonucleotides at a concentration of 200 nM oligonucleotide for four hours in the presence of lipofectin. Results were normalized and expressed as a percent of control. In this initial screen, oligonucleotides giving a reduction of either B-raf mRNA transcript of approximately 30% or greater were considered active. According to this criterion, oligonucleotides 13722, 13724, 13726, 13727, 13728, 13730, 13732, 13733, 13736, 13739, 13740, 13741, 13742, 13743, 14135, 14136, 14138 and 14144 were found to be active. These sequences are therefore preferred. Of these, oligonucleotides 13727, 13730, 13740, 13741, 13743 and 14144 showed 40-50% inhibition of one or both B-raf transcripts in at least one assay. These sequences are therefore more preferred. In one of the two assays, ISIS 14144 (SEQ ID NO: 23) reduced levels of both transcripts by 50-60% and ISIS 13741 (SEQ ID NO: 22) reduced both transcripts by 65-70%. These two sequences are therefore highly preferred.

Dose response experiments were done in both T24 cells and A549 cells for the two most active oligonucleotides, ISIS and ISIS 14144 (SEQ ID NO: 89 and 90), along with mismatch control sequences having 1, 3 or 5 mismatches of the ISIS 13741 sequence. ISIS 13741 and 14144 had almost identical activity in this assay when the upper B-raf transcript was measured, with IC50s between 250 and 300 nM. The mismatch controls had no activity (ISIS 14531) or slight activity, with a maximum inhibition of less than 20% at the 400 nM dose (ISIS 14530, ISIS 14529). Against the lower B-raf transcript, ISIS 13741 and ISIS 14144 had IC50s of approximately 350 and 275 nM, respectively in this assay, with the mismatch controls never achieving 50% inhibition at concentrations up to 400 nM. Therefore, ISIS 13741 and 14144 are preferred.

Reduction of B-raf mRNA levels was measured in T24 cells by these oligonucleotides (all are phosphorothioate oligodeoxynucleotides) after a 4-hour treatment in the presence of lipofectin. Results are normalized to G3PDH and expressed as a percent of control. Against the upper transcript, ISIS 13741 and 14144 were again most active, with IC50s of approximately 100 nM and 275 nM, respectively, in this assay. The mismatch controls 14529 and 14531 had no activity, and the mismatch control 14530 achieved a maximum reduction of raf mRNA of approximately 20% at a 400 nM dose. Against the lower transcript, ISIS 13741 had an IC50 of approximately 100-125 nM and ISIS 14144 had an IC50 of approximately 250 nM in this assay, with the mismatch controls completely inactive. Therefore ISIS 13741 and 14144 are preferred.

2′-Methoxyethoxy (2′-MOB) Oligonucleotides Targeted to B-raf:

The oligonucleotides shown in Table 11 were synthesized. Nucleotides shown in _(bold) are 2′-MOE. 2′-MOE cytosines are all 5-methylcytosines. For backbone linkage, “s” indicates phosphorothioate (P═S) and “o” indicates phosphodiester (P═O).

TABLE 11 2′-MOE oligonucleotides targeted to human B-raf (bold = 2′-MOE) ISIS # Sequence/modification SEQ ID NO: 13741 CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT 89 15339 CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT 89 15340 CoToGoCoCoToGoGoAoToGsGsGSTSGsTsTsTsTsT 89 15341 CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT 89 15342 CoToGoCoCsTsGsGsAsTsGsGsGsTsGoToToToToT 89 15343 CsTsGsCsCsTsGsGsAsToGoGoGoToGoToToToToT  89 15344 CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT  89 These oligonucleotides were tested for their ability to reduce B-raf mRNA levels in T24 cells. Against the lower transcript, ISIS 13741 (P═S deoxy) and ISIS 15344 (P═S deoxy/MOE) had IC50s of approximately 250 nM. The other two compounds tested, ISIS 15341 and 15342, did not achieve 50% inhibition at doses up to 400 nM. Against the upper transcript, ISIS 13741 and 15344 demonstrated IC50s of approximately 150 nM, ISIS 15341 demonstrated an IC50 of approximately 200 nM and ISIS 15342 did not achieve 50% reduction at doses up to 400 nM. Based on these results, ISIS 15341, 13741 and 15344 are preferred.

Example 19 CX-1 Cell Adhesion to TNF-Alpha-Activated Hepatic Sinusoidal Endothelial Cells is Blocked by Pretreatment with Murine C-raf Antisense Oligodeoxynucleotide

CX-1 cells are a highly metastatic, poorly differentiated colorectal carcinoma cell line which produce CEA (what is this?). CX-1 cells can adhere to TNF_(α)-activated murine hepatic sinusoidal endothelial cells in an E-selectin is dependent manner. To determine whether pre-treatment of hepatic endothelial cells with c-raf antisense oligodeoxynucleotide (ODN) could inhibit TNF-_(α) dependent CX-1 cell adhesion, hepatic endothelial cells were treated with different concentrations of c-raf ODN for 4 h, cultured for an additional 48 h, then stimulated or not with 50 ng/ml TNF-alpha for an additional 2 h (for RNA analysis) or 5 h (for adhesion assay). The ODN had the following sequence: 5′-ATGCATTCTGCCCCCAAGGA-3′ (SEQ ID NO: 109), in which the first five and last five nucleotides have 2′-O-methoxyethyl modifications and the internucleoside linkages are all phosphorothioates.

Liver sinusoidal endothelial cells (LSEC) were obtained by perfusion of normal mouse livers with pronase and collagenase, followed by separation of parenchymal and non-parenchymal cells on metrimazide density gradients. The cells were cultured in 24-well plates which were pre-coated with rat tail (type I) collagen for 5-7 days prior to their use in the adhesion assay. Tumor cell adhesion to the endothelial cells was measured as previously described (Brodt et al., Int. J. Cancer 71:612-619, 1997). Briefly, tumor cells were radiolabeled with Na⁵¹Cr and 10⁵ cells were added per well of endothelial cells which had been pre-activated (or not) with 50 ng/ml TNF-_(α) for 5-6 hr. The plates were centrifuged for 10 min at 400 rpm, then incubated at 37° C. for 1 h. Unattached cells were removed by repeated washing, the monolayers lysed with 1N NaOH and readioactivity in the lysates measured using a gamma counter. The total number of endothelial cells per well at the time of the assay was about 2.5×10⁵.

To test the effect of c-raf antisense ODN on tumor cell adhesion to the endothelial cells, the cells were cultured for 5 days, the medium removed and replaced with Opti-MEM medium containing 3 μl lipofectamine (both from Life Technologies, Burlington, Ontario, Canada) with or without different concentrations of the CON. Incubation with the ODN was for 5 h at 37° C. at which time the medium was aspirated, replaced with RPMI containing 10% fetal calf serum (FCS) and the cells incubated at 37° C. for 48 h prior to the adhesion assay.

In response to TNF-_(α), E-selectin mRNA expression in the endothelial cells was significantly higher than in untreated cells. Pretreatment of these cells with c-raf ODN, but not with control ODN, significantly reduced c-raf expression and abolished E-selectin induction in a dose-dependent manner. When adhesion of CX-1 cells to the endothelial cells was subsequently measured, the incremental increase in adhesion due to TNF-_(α) activated E-selectin was reduced in an ODN dose-dependent manner and abolished at a concentration of 100 nM antisense ODN (FIG. 1). Control ODN had no effect on E-selectin expression or tumor adhesion.

Example 20 CX-1 but not MIP-101 Cells Induce Cytokine and E-Selectin Expression Upon Entry into the Hepatic Circulation

Highly metastatic murine carcinoma H-59 cells rapidly induce cytokine and E-selectin expression upon intrasplenic/portal injection in syngeneic mice. The following study tested whether colorectal carcinoma cells that are highly metastatic to the liver could induce a similar host cytokine response when xenotransplanted into nude mice.

Experimental liver metastases were generated by intrasplenic/portal injection of tumor cells as previously described (Long et al., Exp. Cell Res. 238: 116-121, 1998). The mice were anesthetized with an intramuscular injection of 2.2 mg/kg Anased (Novopharm, Toronto, ON), followed by 11 mg/kg Ketalan (Bimeda-MTC, Cambridge, ON). They were then inoculated intrasplenically with 1-2×10⁶ CX-1 cells and splenectomized 1 min later. The mice were sacrificed 4-6 wk later and the liver metastates enumerated immediately, without prior fixation.

Mice received one tail vein injection of 25 mg/kg c-raf antisense or control ODN at 24 h, and a second injection of 6 mg/kg ODN 4 h, prior to the intrasplenic/portal injection of 10⁶ CX-1 cells. Following tumor cell injection, the animals received 1 injection of 6 mg/kg ODN at 4 h and thereafter 1 weekly injection of 25 mg/kg ODN from day 3 onward until the end of the experiment. A second, control group was injected with vehicle (saline) only at the time of ODN injection.

Following the intrasplenic/portal injection of CX-1 cells, there was a rapid increase in hepatic TNF-_(α) (FIG. 2A) and IL-1β mRNA expression. This increase was first detectable at 30 min, reached 10-fold relative to control levels at 4 h and remained high for up to 48 h post tumor inoculation. The increase in cytokine expression was followed by an increase in E-selectin mRNA expression which was measurable at 1 h, reached maximal levels at 4 h and remained high for 48 h post tumor inoculation. The injection of the non-metastatic colorectal carcinoma MIP-101 failed to trigger a cytokine response or E-selectin expression for up to 48 h following tumor cell injection (FIG. 2A-C). A similar E-selectin induction by CX-1 cells was also subsequently confirmed in athymic nude mice (FIG. 2D).

Example 21 Reduction in Tumor-Induced Hepatic E-Selectin Expression Following Treatment with c-raf Antisense ODN

To determine whether a reduction in c-raf levels can inhibit tumor-induced hepatic E-selectin expression, CX-1 cells were injected into nude mice pretreated with c-raf ODN, 24 and 4 h prior to tumor cell inoculation. Livers were harvested 4 h post tumor cell inoculation and c-raf and E-selectin mRNA levels were analyzed using RT-PCR and Northern blotting. Injection of c-raf antisense, but not control ODN, significantly reduced hepatic c-raf and essentially abrogated tumor-induced E-selectin expression.

In brief, total RNA was extracted using the Trizol reagent (Life Technologies, Inc.) and reverse-transcribed in a 20 μl reaction mixture containing 50 mM Tris-HCl, pH 8.3, 30 mM KCl, 8 mM MgCl₂, 1 mM dNTPs and 0.2 units of avian myeloblastosis virus (AMV) reverse transcriptase. In each case, the 3′-antisense oligonucleotide (2 μM) was used to initiate reverse transcription. The mixture was incubated sequentially for 10 min at 25° C., 60 min at 37° C. and 5 min at 95° C. cDNAs were amplified by PCR using the TNF-_(α), IL-10 or E-selectin specific oligonucleotides described (Khatib et al., Cancer Res. 59:1356-161, 1999). For amplification, a total of 25 PCR cycles were performed each consisting of 30 sec at 94° C., 30 sec at 56° C. and 30 sec at 72° C. using a PerkinElmer Life Sciences thermocycler. Amplified PCR products were analyzed on a 1.5% agarose gel. To determine the effect of ODN treatment on tumor-induced E-selectin expression, nu/nu or C57B16 mice were injected i.v. with 25 mg/kg ODN 24 and 4 h prior to the intrasplenic/portal injection of 2×10⁶ CX-1 cells. The livers were removed 4 h following tumor cell injection and the RNA extracted as described. To test the effect of c-raf treatment on TNF-_(α) induced E-selectin expression, the endothelial cells were incubated with 200 nM ODN in Optimem medium for 4 h, the ODN removed, the cells washed and maintained in RPMI medium supplemented with 10% serum for 48 h. Two hours prior to RNA extraction, 50 ng/ml TNF-_(α) were added to the endothelial cell cultures for E-selectin mRNA induction.

Example 22 Treatment with c-raf Antisense ODN Inhibits Experimental Liver Metastasis of CX-1 Cells

To investigate whether reduced E-selectin expression in c-raf antisense ODN treated mice altered the course of experimental liver metastasis, nude mice were tail vein inoculated with c-raf antisense or control ODN 24 and 4 h prior to, as well as 4 h following, the intrasplenic/portal injection of 2×10⁶ CX-1 cells. Maintenance ODN injections were administered once weekly from day 3 onward until the end of the experiment, 4-5 weeks later. In three in vivo experiments performed, the number of metastases in c-raf antisense ODN-treated mice was significantly reduced relative to vehicle or control ODN-treated animals, while no significant difference was observed between the number of metastases in the two control groups. Results of a representative experiment are shown in FIG. 3. The median number of metastases based on pooled data from all three experiments in vehicle treated mice was 24 (range 3-100, n=11), in control ODN treated mice, it was 44 (range 14-100, n=13) and in c-raf antisense ODN treated mice it was 6 (range 2-21, n=20), representing an 86% reduction in the number of metastases relative to control ODN treated mice. Previous experiments have shown that the rodent-specific c-raf antisense ODN does not affect c-raf expression in human cells since the murine c-far ODN sequence used in these studies has no homology to the human sequence.

c-raf antisense ODN had no direct deleterious effect on CX-1 cell growth at concentrations used to block E-selectin expression in endothelial cells as measured by MTT [3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide] assay (Long et al., Exp. Cell Res. 238:116-121, 1998) (FIG. 4). In the MTT assay, Cells were seeded in 24 well plates at a density of 5×10⁴ cells/well and cultured overnight in RPMI containing 110% serum. Different concentrations of c-raf or control ODN were then added and cell viability measured daily for 3 days.

The antisense ODN sequence had no detectable deleterious effect on the human carcinoma CX-1 cell growth in vitro when tested at concentrations which were effective in blocking endothelial E-selectin induction in mouse endothelial cells. Inhibition of the host tumor-induced activation of E-selectin resulted in a marked reduction in the number of experimental liver metastasis. The use of human raf ODNs (A-raf, B-raf or C-raf), particularly ISIS 5142, for prevention and treatment of any type of metastases is within the scope of the present invention. 

What is claimed is:
 1. A method of preventing or treating tumor metastasis in an animal, comprising administering to said animal an oligonucleotide 8 to 50 nucleotides in length which is targeted to mRNA encoding human raf and which is capable of inhibiting raf expression.
 2. The method of claim 1 which is targeted to mRNA encoding human A-raf.
 3. The method of claim 1 which is targeted to mRNA encoding human B-raf.
 4. The method of claim 1 which is targeted to mRNA encoding human c-raf.
 5. The method of claim 4 which is targeted to a translation initiation site, 3′ untranslated region or 5′ untranslated region of mRNA encoding human c-raf.
 6. The method of claim 1 which has at least one phosphorothioate linkage.
 7. The method of claim 1 wherein at least one of the nucleotide units of the oligonucleotide is modified at the 2′ position of the sugar moiety.
 8. The method of claim 7 wherein said modification at the 2′ position of the sugar moiety is a 2′-O-alkyl, a 2′-O-alkyl-O-alkyl or a 2′-fluoro modification.
 9. The method of claim 1, wherein said oligonucleotide is a chimeric oligonucleotide.
 10. The method of claim 1, wherein said metastasis is a liver metastasis. 